The analysis of the size of nucleic acids is useful for many research and diagnostic applications. Electrophoresis, e.g., agarose gel electrophoresis, polyacrylamide gel electrophoresis and capillary electrophoresis, is commonly used for the size analysis of nucleic acids. Mass spectrometry has also been used for size analysis, as nucleic acid fragments of different sizes, such as those produced by a primer extension reaction, have different molecular masses (Ding and Cantor, 2003, Proc Natl Acad Sci USA, 100, 7449-7453).
Below are several examples of the use of size analysis. For example, the presence of a mutation which creates a restriction enzyme site can be detected by treatment with the said enzyme, followed by the analysis of the sizes of the treated products. The presence of shorter fragments of a particular size indicates that the mutation is present. Conversely, the presence of longer DNA fragments corresponding to the unrestricted state is suggestive of the absence of the mutation. If the restriction enzyme used is sensitive to the methylation status of the target DNA fragment, then this type of analysis can also be used for the analysis of DNA methylation. Thus, if an enzyme that only cuts unmethylated DNA is used, then the presence of shorter restricted DNA fragments is indicative of the presence of unmethylated DNA. Conversely, the presence of the longer unrestricted DNA fragments is suggestive of the presence of methylated DNA. The interpretation of these results would be reversed if an enzyme such as McrBC (Sutherland, et al. 1992, J Mol Biol, 225, 327-348), which cuts methylated DNA and which does not cut unmethylated DNA, is used.
As another example, it is known that cell-free fetal DNA in maternal plasma is of a smaller size than maternal DNA (Chan, et al. 2004, Clin Chem, 50, 88-92; Li, et al. 2004, Clin Chem, 50, 1002-1011) (see also European Patent Application No. 03405742.2 “Non-invasive detection of fetal genetic traits”). Thus, size fractionation by electrophoresis has been used to enrich for fetal DNA in maternal plasma (Li, et al. 2005, JAMA, 293, 843-849).
In the field of oncology, increased DNA integrity has been observed in cancer patients (Hanley, et al. 2006, Clin Cancer Res, 12, 4569-4574; Jiang, et al. 2006, Int J Cancer, 119, 2673-2676; Umetani, et al. 2006, J Clin Oncol, 24, 4270-4276; Wang, et al. 2003, Cancer Res, 63, 3966-3968) (see also U.S. Pat. No. 6,964,846). This phenomenon is thought to be related to necrotic changes which are associated with the tumor. DNA integrity in cancer patients has been analyzed by separate real-time PCR assays for different sized amplicons. Exact Sciences also has a proprietary DNA integrity assay (for more information see the web site exactsciences.com/applied/applied.html).
DNA size analysis has also been used for the analysis of viral-derived nucleic acid sequences, such as the size of Epstein-Barr virus (EBV) DNA in the plasma of patients with nasopharyngeal carcinoma and certain lymphomas (Chan, et al. 2003, Cancer Res, 63, 2028-2032). Size analysis has also been used for the measurement of RNA integrity (Wong, et al. 2006, Clin Cancer Res, 12, 2512-2516; Wong, et al. 2005, Clin Chem, 51, 1786-1795). Such analysis might be of use in clinical diagnosis, as decreased RNA integrity has been observed in cancer patients. Also, placental RNA in the plasma of pregnant women has been shown to be consisted of partially degraded fragments, with a 5′ preponderance (Wong, et al. 2005, Clin Chem, 51, 1786-1795). It has been suggested that oxidative stress would decrease the integrity of such placental-derived mRNA (Rusterholz, et al. 2007, Fetal Diagn Ther, 22, 313-317). Digital PCR followed by DNA sequencing has been used for the analysis of the size distribution of plasma DNA in patients with colorectal tumors (Diehl, et al. 2005, Proc Natl Acad Sci USA, 102, 16368-16373).
The present invention provides novel methods for analyzing the size of nucleic acids, especially nucleic acids derived from the same longer sequence, and the relative abundance of such nucleic acids of different lengths in a test sample.